Morphological and histochemical observations of the organic components of ostrich eggshell

The organic component of the avian eggshell can be divided into 3 portions, the shell membranes, the matrix and the cuticle. These have been well characterised in the chicken but little has been published with regard to the ostrich (Struthio camelus). A number of recent studies have indicated that the cause of intra-shell embryonic deaths in the ostrich is similar to intra-shell embryonic deaths that occur in the chicken. These deaths in the chicken are associated with the loss of or damage to the waxy cuticle and other organic components of the eggshell, which is reported to be absent in the ostrich eggshell. In this study, preliminary morphological and histochemical analyses, at the level of the light and electron microscope, have characterised the various organic components of the ostrich eggshell. The results of the histochemical and electron microscopical analyses suggest that there may only be 1 shell membrane in this species, which could play a major role in the limitation of bacterial penetration to the embryonic chamber The shell membrane has a distinct elemental profile as determined by EDS analysis. The matrix is shown to decrease in mesh size from the mammillary layer to the vertical crystal layer. The closer packing of the mesh may indicate the presence of a morphologically discernible termination signal to calcification or the remnants of an evolutionary calcified cuticle. The matrix of the pores may also form a defensive barrier against bacterial invasion, which could be damaged as a result of dipping the eggs before incubation.     

Morphology of the distal tip of the upper mandible of the ostrich ( Struthio camelus ) embryo during hatching

1. The morphology of the distal part of the upper mandible in ostrich embryos was investigated using scanning electron microscopy just prior to and during hatching. 2. Although a keel-like structure on the tip of the upper mandible superficially resembles an egg tooth it appears to play no role during hatching. 3. The distal tip of the upper mandible is covered by an amorphous layer, the right side of which disappears during the hatching process. This layer acts to protect the beak during the rubbing process which creates a hole in the inner shell membrane during hatching.     

鸡蛋壳的超微结构研究

本研究采用高分辨率的扫描电镜观察海赛克斯鸡蛋壳的超微结构,详细分析了其结构模式。蛋壳从内到外包括壳膜、锥体层、柱状层、表面晶体层、覆盖层五部分。同时研究结果表明,鸡蛋壳强度的大小可能与壳膜层壳膜的厚度,壳膜纤维的粗细,锥体层乳头间的空隙大小以及覆盖层上裂隙的深浅和数量有关。     

Transmission electron microscopy of the vertical crystal layer and cuticle of the eggshell of the domestic fowl

1. Eggshell pieces (1 cm2) were decalcified using a solution of EDTA (200 g/litre, pH 6.9 to 7.0) in paraformaldehyde (2 g/litre) and glutaraldehyde (0.5 g/litre) in phosphate buffer. 2. They were prepared for transmission electron microscopy (TEM). 3. TEM identified a vertically aligned matrix associated with the vertical crystal layer (VCL). It is hypothesised that the vertical orientation of calcite crystals in the VCL is closely linked to this vertical matrix. 4. TEM also revealed the presence of a 2-layered cuticle, the inner layer containing vesicles which were absent in the outer. 5. Cuticular vesicles contain hydroxyapatite and are thought to play a role in the termination of shell formation. The current paper presents data relating to microbial apatitic systems that strengthen this hypothesis.     

雏鸵鸟睾丸的结构及睾丸内神经肽Y的表达

采用光镜和电镜技术,观察雏鸵鸟睾丸的显微和超微结构;采用免疫组织化学方法对NPY在睾丸内的表达进行研究,探明NPY在睾丸内的表达规律.光镜下可见,雏鸵鸟睾丸表面有一层由坚韧的结缔组织构成的白膜,它向睾丸实质内形成的结缔组织不发达,不形成睾丸纵隔和睾丸小隔;雏鸵鸟的曲精小管直径为85～90 μm,生精上皮由支持细胞和1～2层生精细胞构成,此时精原细胞已开始分裂增殖,可见明显的分裂相,初级精母细胞数量少,体积大.电镜下可见,精原细胞胞质内有少量线粒体、游离核糖体和内质网;初级精母细胞富含线粒体、游离核糖体、内质网和溶酶体;支持细胞与精原细胞之间界限清晰,胞质内含较多游离核糖体,其他细胞器不发达;间质细胞的胞核形状不规则,内含较多脂滴.免疫组化显示,NPY在曲精小管周围、睾丸小血管周围、精母细胞和睾丸间质内均有表达,在睾丸小血管周围和精母细胞内表达量较高,而在曲精小管周围和睾丸间质内表达量较低.     

鸵鸟精子的超微结构

为了丰富鸵鸟精子的形态资料和为提高鸵鸟的人工授精技术提供参考,采用透射电镜和扫描电镜对鸵鸟精子的超微结构进行观察,并且与其它非雀形目鸟类以及哺乳动物的精子结构进行了比较。结果表明,鸵鸟精子呈细长的圆柱状,分为头部、颈段、中段、尾段、末段,其结构与美洲鸵和Tinamou精子相似。鸵鸟精子头部由顶体和精子核组成,精子核中有一条顶体刺。鸵鸟精子没有明显的颈部。中段的外周环绕着线粒体鞘。主段的中央为轴丝,轴丝外周有纤维鞘。鸵鸟精子的超微结构与美洲鸵和tinamou相似,但是与其它非雀形目的鸟类和哺乳动物的精子有很大的区别。     

Ultrastructural features of the follicular wall in developing follicles of the sexually immature ostrich (Struthio camelus)

The ultrastructure of the follicular wall in primordial, previtellogenic and vitellogenic follicles of the sexually immature ostrich is described in the present study. The follicular wall consists of a zona radiata, granulosa cell layer, basal lamina and thecal layer. Cytoplasmic processes from the plasma membranes of the granulosa cell layer and the ovocyte form the zona radiata in previtellogenic and vitellogenic follicles. The granulosa cell layer transforms from simple cuboidal epithelium in primordial follicles to simple columnar or pseudostratified columnar epithelium in previtellogenic and vitellogenic follicles. Transosomes were observed along the apical and lateral plasma membranes of granulosa cells. The thecal layer in previtellogenic and vitellogenic follicles consists of interna and externa components. The fibroblasts in the theca externa contain microfilaments, which are thought to be actin filaments. The study revealed ultrastructural features, which are associated with the transportation of yolk precursors and nutrients into the ovoplasm. In addition, the study indicates that, although the cells in the theca externa contain microfilaments, they do not possess the ultrastructural characteristics of smooth muscle cells.     

A study of the progressive deposition of shell in the shell gland of the domestic hen

It is known that the organic matrix of the shell is not distributed evenly throughout the calcified portion and an experiment has been described in which incompletely shelled eggs were removed from a group of domestic hens after being in the shell gland for various known periods.

Rate of shell deposition and the timing of the deposition of the various layers in the shell has thus been determined, and it was found that shell deposition was initially slow for the first 3 h in the gland and thereafter more rapid, at a constant rate of 322 mg h^?1, until termination after 17 or 18 h. The narrow layer low in matrix at the top of the mammillary layer of the shell was laid down during the 7th to 9th hour in the gland and did not appear to be associated with any change in shell deposition rate. It was also found that the characteristic pole to pole thickness patterns of shells were present at the earliest measurable stage, that is after about 8 h in the gland.     

Identification of the major proteins of the organic matrix of emu (Dromaius novaehollandiae) and rhea (Rhea americana) eggshell calcified layer

1. The eggshell is a composite consisting of 95% calcite and an organic matrix. 2. While many proteins of the chicken eggshell matrix have already been identified, little is known about the matrix of other birds. 3. Isolation of the emu and rhea eggshell matrix and analysis of its major constituents showed that the predominant components were C-type lectin-like proteins related to those of ostrich, chicken and goose. 4. Serum albumin, vitelline membrane outer layer protein I (VMO-I) and the turpentine-induced acute phase serum protein 18-B were identified as minor components of the emu shell matrix. Both eggshell matrices also contained a novel proline- and alanine-rich protein. 5. Like ostrich, and unlike chicken and goose, both emu and rhea eggshell matrix contained two different C-type lectin-like proteins as major components, indicating that the occurrence of two proteins of this family may be widespread among ratites.     

Macro-microscopic study on the toepad of ostrich (Struthio camelus)

The ostrich foot has four toepads, two on the 3rd digit, one on the 4th digit and one at metatarso-phalangeal joint. Previous studies have not detailed the histo-morphological structure of these toepads. In this study, we have described the macroscopic and microscopic structures of the toepad of ostrich (Struthio camelus). Numerous papillae with different direction, length and thickness have been observed grossly on the ventral surface of each toepad. Histological examinations have revealed that the epidermis of the ostrich toepad, similar to other digitigrades, consists of an outer stratum corneum and an inner stratum germinativum (which is subdivided into basal, intermediate and transitional layers). The stratum corneum has several layers of flattened horny cells. The nuclei of basal cells have several mitotic figures. The cytoplasm of the stratum germinativum cells has multiple lipid droplets and multigranular bodies (in transitional cells only). Scanning electron microscopic examination revealed presence of collagen fibers in mid and deep dermis of each toepad. These fibers run parallel and connect to each other by very thin fibrils which are branched, crossed with each other in an oblique direction. Such arrangement of these collagen fibers, thin fibrils and presence of digital cushion are likely to be responsible for the protection of the underlying soft tissues and absorption of concussion.     

雄性雏鸵鸟生殖器官的形态学研究

为了探明雄性雏鸵鸟生殖器官的形态学特点,采用石蜡切片和HE染色技术,对6羽45日龄雄性非洲雏鸵鸟的生殖器官进行了研究,并与家禽的相关结构进行了比较。在光镜下观察了雄雏鸟生殖器官的形态结构,结果表明:(1)鸵鸟睾丸没有睾丸纵隔和睾丸小叶,曲精小管中精原细胞排列紧密且整齐,睾丸间质较少;(2)附睾由睾丸网、输出小管和附睾管组成;(3)输精管管壁由黏膜、肌层和外膜3层组成;(4)阴茎由2个圆形的纤维性淋巴体、淋巴间隙和微血管共同构成。     

高邮鸭蛋壳超微结构观察

高邮鸭蛋壳的扫描电镜观察表明,由内向外可将蛋壳分为壳膜层、乳锥层、柱状层、表面晶体层和表层。青壳蛋与白壳蛋在超微结构上存在显著差异,从而导致青壳蛋的蛋壳强度明显高于白壳蛋。     

Molecular identification of encephalitozoon hellem in an ostrich

Microsporidia are obligate, intracellular, eukaryotic parasites found in a wide variety of vertebrate and invertebrate hosts. Scientific literature contains a small number of reports of these parasites in psittacine hosts, and recently microsporidiosis was reported in the first nonpsittacine host, an ostrich. DNA was extracted from formalin-fixed, paraffin-embedded ostrich tissues, and a portion of the small subunit ribosomal RNA gene was sequenced to identify the microsporidian species. The organisms were identified as Encephalitozoon hellem, a parasite species that was first described in immunocompromised humans and recently reported in three psittacine species.     

Genetic parameters for eggshell traits in ostriches

1. A study was conducted on ～14000 ostrich eggs to estimate genetic parameters for eggshell traits that could benefit the hatchability of ostrich eggs. Traits measured included the number of pores on the eggshell, the average diameter of these pores, the total area of pores on the eggshell, permeability (pore area/shell thickness) and eggshell thickness.

2. Heritability estimates ranged from 0·16 for total pore area to 0·41 for the natural logarithm of pore count. The heritability estimates for water loss on 21 and 35?d (WL21 and WL35) of incubation were high at 0·23 and 0·24, respectively.

3. On a genetic level, pore count was negatively correlated with average pore diameter (?0·73) and shell thickness (?0·28), whereas it was positively correlated with total pore area (0·58), WL21 (0·24) and WL35 (0·34). The direct and maternal genetic correlations of pore count with total pore area (0·58) and permeability (0·59) were high and significant. Permeability was positively correlated to WL21 and WL35, both on the direct and maternal genetic levels.

4. The estimated genetic parameters indicate that it should be possible to select for the various eggshell traits in ostrich eggs, or for permeability and water loss. However, as a trait with an intermediate optimum, direct selection for permeability and other eggshell traits would not be straightforward, and the possible application of these results to improve hatchability of ostrich eggs in the future needs consideration.     

The use of the plasma chemistry unit as an aid to the scanning electron microscope study of avian egg‐shell structure

1. In order to study the mammillary layer of the avian egg shell by scanning electron microscopy, it is necessary to separate the outer shell membrane from the calcified shell.

2. Chemical methods of effecting membrane removal are difficult to standardise due to variations in the strength of the membrane‐shell bond.

3. The use of reactive gas plasma provides an alternative, more efficient method for removing membranes without the risk of damage to underlying crystalline structures.     

Eggshell strength: A relationship between the mechanism of failure and the ultrastructural organisation of the mammillary layer

1. Stress analysis using modern computer modelling techniques indicated that the shell should fail in tension as a result of the accumulation of stress at its inner most surface with the characteristic notched appearance of the mammillary layer providing the ideal crack initiation site.

2. Practical observations however indicated that crack initiation does not inevitably lead to catastrophic failure. This partly explains why previous attempts to predict shell failure from classical engineering theory underestimated the actual force required to break an egg.

3. Crack initiation should now be denned as localised trauma, characterised by a series of radial cracks which propagate outwards from the load point at first in a stable manner. As the interaction between the shell and its loading environment continues, one or more of these minor cracks become unstable, thus giving rise to catastrophic failure.

4. The fracture toughness of the eggshell is a measure of its resistance to unstable crack growth and in this respect there appears to be a link between the latter and the structural organisation of the mammillary layer.     

Categorisation and causes of abnormal egg shells: relationship with stress

Concern regarding the proportion of brown eggs being laid with abnormal egg shells led to an investigation of the relationship between disturbance or stress and egg shell appearance and quality. The abnormalities fell into two main classes: misshapen or bulging eggs and eggs coated with a superficial layer of amorphous calcium, variously termed dusted, white banded, chalky or pink eggs. Translocation of hens from pens to cages resulted in a decrease in egg production and an increase in the proportion of abnormal eggs over the succeeding 18 d. Disturbance to flocks on deep litter resulted in an increase in the proportion of eggs laid with abnormal shells on the following day, not only in the flocks directly affected but also in an adjacent flock. Exclusion from their nests of birds accustomed to laying in nest boxes resulted in disturbed prelaying behaviour, retention of eggs in the shell gland and an increased proportion of coated eggs. Administration of 0.1, 0.25 or 1.0 mg of adrenaline subcutaneously resulted in the retention of eggs currently in the hens' shell glands and in an increased proportion of eggs with abnormal egg shells being laid during the following 10 d. A dose response effect was apparent. There was evidence that the nature of the particular abnormality produced was dependent on the stage of egg formation at which the disturbance was imposed and, if retention followed, upon the length of time the egg was retained. Disturbances when eggs were only lightly calcified tended to result in misshapen eggs, while those occurring when oviposition was imminent tended to result in coated eggs. Dusted or pink eggs followed moderate retention whereas white banded eggs were seen after prolonged retention. These observations may provide a basis for a non-invasive method of assessing stress in laying hens as well as helping to account for hitherto inexplicable occurrences of declining egg shell quality.     

The structure of the shell and polar plugs of the egg of the whipworm, Trichuris trichiura (Nematoda: Trichuridae) from the Samango monkey (Cercopithecus albogularis)

The structure of the shell of the egg of Trichuris trichiura was examined using light microscopy as well as scanning and transmission electron microscopy. The results confirmed its three-layered structure and provided evidence that the cores of the polar plugs, which could be seen to be extensions of the shell's middle layer, could be lost en bloc, either mechanically or chemically, and in this way probably provided an exit for the first stage larva at hatching.     

Immunoreactivities to protein gene product 9.5, neurofilament protein and neuron specific enolase in nerves in the oviduct of the sexually immature ostrich, Struthio camelus

The present study investigated the distribution of nerves in the oviduct of the sexually immature ostrich. The presence of protein gene product 9.5, neurofilament protein and neuron specific enolase nerve fibres were demonstrated in the infundibulum, magnum, isthmus, shell gland and vagina. Nerve fibres containing protein gene product 9.5, neuron specific enolase and neurofilament protein were particularly numerous in the tunica muscularis and intermuscular connective tissue areas of the shell gland and vagina. The presence of a large number of nerves in these oviductal regions is probably important in the coordination of muscle contraction. An interesting finding of the study was the presence of protein gene product 9.5 and neuron specific enolase immunopositive nerve fibres in the walls of blood vessels. It is likely that these nerves are autonomicin nature and play a role in the regulation of blood flow. This study has shown the presence of an extensive neural network in the oviduct of the ostrich. In addition, the results of the investigation have shown that the neuronal markers protein gene product 9.5, neurofilament protein and neuron specific enolase can be used to demonstate nerve fibres in the ostrich.     

Age-related morphometry of equine calcified cartilage

Although there are many studies in the equine literature focused on articular diseases and the aetiology of osteoarthritis, few have concentrated on normal articular structures and how they change with age. The objective of this investigation was to study the thickness and morphology of the calcified cartilage layer of the distal metacarpus over a range of ages. A parasagittal slab of bone was sectioned from the region of sesamoid contact on the medial condyle of the metacarpi from 34 horses. The slab of bone was preserved, dehydrated and embedded, undecalcified, in methylmethacrylate and then stained with toluidine blue. Six repeatable fields of interest from the distal aspect of each metacarpus were digitised and examined to determine the morphology of the calcified cartilage layer. The thickness of the calcified cartilage, range 88-426 microm, was estimated using a method of integration. The results indicate an age-related influence on the thickness of the calcified cartilage layer, generally increased in older horses. While this finding is significant, perhaps more importantly a positional relationship was also identified, indicating that pressures endured by different regions within a joint may dictate morphological development of the tissues. This study has begun to lay the groundwork to determine whether the calcified layer of the hyaline cartilage could be involved in the development of osteoarthritis.